The photoreceptor rod outer segment cGMP phosphodiesterase (PDE) converts cytoplasmic 3',5'-cGMP to 5'-GMP in a light-dependent manner, acting as a link in the amplification cascade of visual transduction. PDE is a complex heterotrimeric enzyme whose cyclic GMP hydrolytic activity is modulated by activators and inhibitors. Molecular mechanisms of activation, inhibition, and interaction between subunits are currently not well understood. By molecular cloning, at least two additional retinal PDE isozymes are predicted to be present in the retina. Their sequences are identical to the alpha subunit except for deviating C-terminal ends. The origin and cellular location of the PDE isozymes is unknown. The proposed research is directed 1) toward an understanding of the gene structure of the PDE alpha-subunit and its predicted isozymes, and 2) toward a definition of functional domains that are involved in interaction with regulators. Initial Southern blotting experiments indicate that the gene encoding the alpha subunit may be very large, and that the PDE isozymes may originate from a single gene by alternative splicing. To initiate gene characterization, we propose to isolate overlapping genomic fragments, to determine the exon/intron arrangement by blotting and DNA sequencing, and to analyze the number of transcripts by RNA blotting. To identify functional domains, we propose to overproduce the subunits of interest in a heterologous expression system, and to reconstitute a functional enzyme. To map functional domains, we propose to initially identify exposed antigenic determinants by screening epitope libraries of individual full-length subunit cDNAs with specific monoclonal antibodies. Peptides comprising identified epitopes will then be synthesized and assayed for competition in activation or inhibition of PDE activity.